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[Preprint]. 2025 Feb 18:2024.05.01.592016.
doi: 10.1101/2024.05.01.592016.

VGLL2 and TEAD1 fusion proteins drive YAP/TAZ-independent tumorigenesis by engaging p300

Susu Guo  1 , Xiaodi Hu  2 , Jennifer L Cotton  2 , Lifang Ma  1 , Qi Li  2   3 , Jiangtao Cui  1 , Yongjie Wang  1 , Ritesh P Thakare  2 , Zhipeng Tao  4 , Y Tony Ip  3 , Xu Wu  4 , Jiayi Wang  1 , Junhao Mao  2
Affiliations

VGLL2 and TEAD1 fusion proteins drive YAP/TAZ-independent tumorigenesis by engaging p300

Susu Guo et al. bioRxiv. .

Update in

Abstract

Studies on Hippo pathway regulation of tumorigenesis largely center on YAP and TAZ, the transcriptional co-regulators of TEAD. Here, we present an oncogenic mechanism involving VGLL and TEAD fusions that is Hippo pathway-related but YAP/TAZ-independent. We characterize two recurrent fusions, VGLL2-NCOA2 and TEAD1-NCOA2, recently identified in spindle cell rhabdomyosarcoma. We demonstrate that in contrast to VGLL2 and TEAD1, the fusion proteins are strong activators of TEAD-dependent transcription, and their function does not require YAP/TAZ. Furthermore, we identify that VGLL2 and TEAD1 fusions engage specific epigenetic regulation by recruiting histone acetyltransferase p300 to control TEAD-mediated transcriptional and epigenetic landscapes. We showed that small molecule p300 inhibition can suppress fusion proteins-induced oncogenic transformation both in vitro and in vivo. Overall, our study reveals a molecular basis for VGLL involvement in cancer and provides a framework for targeting tumors carrying VGLL, TEAD, or NCOA translocations.

Keywords: Hippo signaling pathway; NCOA2; TEAD1; VGLL2; fusion; p300.

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Conflict of interest statement

Disclosure and competing interests statement Dr. Xu Wu has a financial interest in Tasca Therapeutics, which is developing small molecule modulators of TEAD palmitoylation and transcription factors. Dr. Wu’s interests were reviewed and are managed by Mass General Hospital, and Mass General Brigham in accordance with their conflict of interest polices.

Figures

Figure 1.
Figure 1.. VGLL2-NCOA2 and TEAD1-NCOA2 Induce TEAD-Mediated Transcriptional Activation.
A. Schematic representation of protein structure of VGLL2, TEAD1, NCOA2, VGLL2-NCOA2 and TEAD1-NCOA2. Tondu motif (TDU), TEA DNA binding domain (TEA), YAP binding domain (YBD), basic Helix-Loop-Helix (bHLH), Per-Arnt-Sim domain (PAS), nuclear receptor interaction domain (NID), transcriptional activation domain (TAD). Arrows point to the break points. B. Immunoblot analysis of YAP5SA, VGLL2-NCOA2, TEAD1-NCOA2, TAZ4SA, TEAD1, VGLL2 and NCOA2 in HEK293T cells transfected with the expression constructs carrying the HA tag. The figure shows the representative results of three biological replicates. C. YAP5SA, VGLL2-NCOA2, TEAD1-NCOA2, TAZ4SA, TEAD1, VGLL2, and NCOA2 induce transcriptional activation of TBS (TEAD binding site)-luciferase reporter (TBS-Luc) in HEK293T cells. n = 3. ****, p < 0.0001. D. Heatmap showing expression levels of the core genes including CTGF, CYR61, ANKRD1 and AMOTL2 significantly regulated in HEK293T cells expressing YAP5SA, VGLL2-NCOA2 and TEAD1-NCOA2. n=3 E. mRNA levels of CTGF, ANKRD1 and CYR61 in HEK293T cells expressing YAP5SA, VGLL2-NCOA2, TEAD1-NCOA2, TEAD1, VGLL2 or NCOA2. n=3; ****, p < 0.0001. NS, no significance.
Figure 2.
Figure 2.. VGLL2-NCOA2 and TEAD1-NCOA2-induced transcription does not require YAP and TAZ.
A. Co-IP assay showing VGLL2-NCOA2 binding to TEAD1 but not YAP5SA. YAP5SA-Flag or TEAD1-Flag was co-expressed with VGLL2-NCOA2-HA in HEK293T cells and immunoprecipitated with an anti-HA antibody. B. VGLL2-NCOA2 binds to endogenous TEAD but not YAP/TAZ. Endogenous YAP/TAZ and TEAD proteins in HEK293T cells were detected by anti-YAP/TAZ and panTEAD antibodies, respectively. C. Co-IP assay showing endogenous YAP/TAZ binding to TEAD1 but not TEAD1-NCOA2. TEAD1-Flag or TEAD1-NCOA2-Flag was expressed in HEK293T cells and immunoprecipitated with an anti-Flag antibody. Endogenous YAP/TAZ proteins were detected by anti-YAP/TAZ antibodies. D. The activity of TBS-Luc reporter in HEK293T cells expressing YAP5SA, VGLL2-NCOA2, or TEAD1-NCOA2, with TEAD inhibitor CP1 (5 μM) treatment or co-expression of TEAD-ENR repressor construct. n=3; ****, p < 0.0001. NS, no significance. E. Schematic representation of TEAD-ENR. TEA DNA-binding domain (TEA) and Engrailed repressor domain (ENR). F. Immunoblot analysis of TEAD-ENR expression in HEK293T cells. G. Co-IP assay showing YAP/TAZ were not essential for VGLL2-NCOA2 binding to endogenous TEAD. VGLL2-NCOA2-HA was expressed in HEK293T cells with or without YAP/TAZ knockdown and immunoprecipitated with an anti-HA antibody. H. Relative mRNA levels of CTGF, ANKRD1, and CYR61 in HEK293T cells with YAP/TAZ knockdown and expressing VGLL2-NCOA2 or TEAD1-NCOA2. n=3; ****, p < 0.0001.
Figure 3.
Figure 3.. Characterization of VGLL2-NCOA2- and YAP- controlled transcriptional and chromatin landscapes.
A. Intersection of ATAC-seq (n=2), RNA-seq (n=3), and CUT&RUN (n=2) datasets in HEK293T cells expressing VGLL2-NCOA2 or YAP5SA. B. Venn diagrams showing the overlaps of ATAC-seq peaks, CUT&RUN peaks, and differentially regulated genes from RNA-seq in HEK293T cells expressing VGLL2-NCOA2 or YAP5SA. C. KEGG pathway enrichment analysis of ATAC-seq peaks identified in HEK293T cells expressing VGLL2-NCOA2 or YAP5SA. The “Hippo signaling pathway” is highlighted in red. D. Distribution of CUT&RUN binding sites for VGLL2-NCOA2 and YAP5SA. E. Motif enrichment analysis of VGLL2-NCOA2 and YAP5SA CUT&RUN Peaks. F. Genomic tracks showing VGLL2-NCOA2 and YAP5SA occupancy at the CTGF, ANKRD1, and CYR61 loci.
Figure 4.
Figure 4.. VGLL2-NCOA2 and TEAD1-NCOA2 engage p300 epigenetic regulators.
A. Diagram showing the BioID proteomic analyses of BirA*-VGLL2-NCOA2, BirA*-TEAD1-NCOA2, BirA*-YAP5SA, and BirA*-TAZ4SA. B. Co-IP assay showing endogenous p300 binding to VGLL2-NCOA2 and TEAD1-NCOA2 but not YAP5SA. C. KEGG enrichment analysis of p300 CUT&RUN peaks in control HEK293T cells and HEK293T cells expressing VGLL2-NCOA2 or TEAD1-NCOA2. The “Hippo signaling pathway” is highlighted in red. D. Motif enrichment analysis of p300 CUT&RUN peaks in control HEK293T cells and HEK293T cells expressing VGLL2-NCOA2 or TEAD1-NCOA2. E. Genomic tracks showing p300 occupancy at the CYR61, ANKRD1, CTGF, and CREB3 loci in control HEK293T cells and HEK293T cells expressing VGLL2-NCOA2 or TEAD1-NCOA2.
Figure 5.
Figure 5.. p300 is required for VGLL2-NCOA2- and TEAD1-NCOA2-induced tumorigenesis in vitro.
A. Co-IP assay showing the NCOA2 fusion part of VGLL2-NCOA2 was essential for p300 binding. VGLL2-NCOA2-V5，VGLL2-NCOA2ΔNCOA2-V5 and VGLL2-NCOA2ΔVGLL2-V5 was expressed in HEK293T cells and immunoprecipitated with an anti-V5 antibody. Endogenous p300 proteins were detected by anti-p300 antibody. B-D. mRNA levels of Ctgf, Ankrd1, and Cyr61 in C2C12 cells expressing YAP5SA, VGLL2-NCOA2, or TEAD1-NCOA2 with or without treatment of A485 (5 μM). n=3; ****, p < 0.0001. NS, no significance. E. Representative image of colony formation of C2C12 cells expressing YAP5SA, VGLL2-NCOA2, or TEAD1-NCOA2 with or without treatment of A485 (5 μM) for 2 weeks. Scale bars, 100 μm. F-G. Colony size (F) and number of colonies (G) formed by C2C12 cells expressing YAP5SA, VGLL2-NCOA2, or TEAD1-NCOA2 with or without treatment of A485 (5 μM). **, p < 0.01. ****, p < 0.0001. NS, no significance.
Figure 6.
Figure 6.. p300 is essential for VGLL2-NCOA2- and TEAD1-NCOA2-induced tumorigenesis in vivo.
A. Representative H&E and IHC staining of Desmin and Ki67 in C2C12-control allograft, C2C12-VGLL2-NCOA2 tumor allograft, and C2C12-TEAD1-NCOA2 tumor allograft. Scale bars, 200 μm. B. Immunoblot analysis of VGLL2-NCOA2-FLAG and TEAD1-NCOA2-FLAG expression in C2C12 cells, detected by an anti-FLAG antibody. C-D. Allograft leg volume of C2C12-VGLL2-NCOA2 (C) and C2C12-TEAD1-NCOA2 (D) after intramuscular injection into the leg of Nude mice with or without intraperitoneal injection of A485 (100 mg/kg). The error bars represent the mean leg volume ± SEM. n = 6. ****, p < 0.0001. E. Representative H&E and IHC staining of Ki67 in C2C12-VGLL2-NCOA2 and C2C12-TEAD1-NCOA2 tumor allografts with or without A485 (100 mg/kg) treatment. Scale bars, 200 μm. F. Percentage of Ki67-positive cells in Figure 6E. n=6; ***, p < 0.001. G-H. mRNA levels of Ctgf, Ankrd1, and Cyr61 in C2C12-VGLL2-NCOA2 and C2C12-TEAD1-NCOA2 tumor allografts with or without A485 (100 mg/kg) treatment. **, p < 0.01. ***, p < 0.001. ****, p < 0.0001.
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